Decoding of the entire base sequence of human genome is ended and decoding of genomic base sequences of other living organisms is under rapid progress. Under these circumstances, vast amounts of base sequence data are accumulating. Gene-related technologies are expected to dramatically develop in a wide range of fields including various disease diagnosis, drug development, breeding of agricultural crops, etc., as the functions of genes in vivo are elucidated on the basis of the genomic base sequence data.
In addition to the base sequence data, functional information of genes also forms the foundation for development of these new fields. As a technology for conducting functional analysis of genes on a large scale that enables development of gene examination, there have been developed electrophoresis, DNA chips and DNA microarrays (hereinafter generally referred to as DNA microarrays), and pyrosequencing, for example. Electrophoresis systems are distributed by Applied Biosystems and Agilent, for example. DNA microarrays are developed by Affymetrix and Nanogen, for example.
However, most of the existing electrophoresis systems and DNA microarrays are based on a fundamental principle of fluorescent detection; thus, it is necessary to give samples fluorescent labeling. This requires lasers and complex optical systems, which causes the system to be large and expensive. In particular, in the field of medicine, single nucleotide polymorphism (SNP) must be detected easily but with high accuracy in order to realize tailor-made medicine.
Most DNA microarrays now under development is based on a fundamental principle of detecting double-stranded DNAs by hybridization; thus, the selectivity of reaction is not very high and the accuracy needs improvement. Moreover, the base sequence analysis based on electrophoresis involves complicated preparation of samples and requires large equipment since high voltage and optical detection units are usually required. Thus, a technology that can satisfy the size, simplicity, economy, and high accuracy requirements has been demanded.
As means to overcome these problems, there are several reports of DNA microarrays of an electric current-detector type combined with redox labeling. Clinical Micro Sensors has developed a technology of detecting a target gene in which one end of a molecule called “molecular wire” is immobilized on a metal electrode and the other end is connected to a nucleic acid probe so that exchange of electrons between the metal electrode and the redox label based on the hybridization with the target gene is detected as a change in electric current (Nonpatent document 1).
TUM Laboratories has developed a technology of detecting hybridization through measuring the redox current at the metal electrode using ferrocenylnaphthalene diimide as a labeling agent having an electrochemical activity (Nonpatent document 2). Toshiba has developed a system of examining medicinal benefits for hepatitis C using DNA chips of a current-detecting type (Nonpatent document 3). According to this technology, neither expensive lasers nor complicated optical systems are required; thus, a small, simple system can be constructed. However, since its fundamental principle is to detect the oxidation-reduction reaction on the metal electrode, current caused by oxidation or reduction will flow if an oxidizing substance or a reducing substance (e.g., ascorbic acid) is present in the sample. This hinders gene detection and decreases detection accuracy. Moreover, along with the current measurement, electrode reaction progresses on the metal electrode. Since the electrode reaction is irreversible and non-equilibrium, corrosion of the electrode, generation of gasses, separation of immobilized nucleic acid, and degradation of stability of current measurement would result. Thus, detection accuracy is degraded especially when measurement is repeated.
There is also a report of an attempt to detect hybridization of DNAs using field-effect devices (Nonpatent document 4). This technology, which is based on the fact that DNA molecules have negative charges in solutions, detects change in electric charge caused by hybridization using the field effect. However, DNA probes formed on a substrate are inherently negatively charged; thus, the change in charge by hybridization with target DNAs is so small that it cannot be distinguished from nonspecific adsorption. Thus, in order for the technology to be suitable for gene examination, improvements in sensitivity and accuracy are necessary. Furthermore while detecting a minute difference (a difference of one base) between two genes is required in the case of single nucleotide polymorphism (SNP), the sensitivity and accuracy (selectivity) of this technology are unsatisfactory and detection is difficult. Moreover, according to a method based on a fundamental principle of hybridization only as in the case of DNA microarrays described above, it is impossible to analyze base sequence of target genes (DNA sequencing).
Electrophoresis technology is popular as a method for conducting DNA sequencing. A system for analyzing short sequences (short sequencing) downsized by forming migration paths on a glass or polymeric plate has been developed (Nonpatent document 5). However, the system requires application of high voltage and an optical system for fluorescence detection and is not different from conventional electrophoresis regarding the fundamental principle. Thus, problems of size, simplicity, economy, etc., still exist.
On the other hand, pyrosequencing is a detection technology utilizing chemical emission from enzymes caused by release of pyrophosphate that accompanies DNA elongation reaction. According to this technology, enzymes and reagents (dATP, dGTP, dCTP, and dTTP) are sequentially added and emission is detected to analyze the base sequences of DNAs. This method is relatively simple and suitable for small-size systems; however, the chemical reaction system is complex and parallel analysis of a large number of genes different from one another is difficult (Nonpatent document 6).
Several DNA detecting sensors utilizing the field effect have been reported. Eagle Research and Development, LLC forms micropores in a silicon substrate and fabricated a gate portion of a field-effect transistor on the inner sidewall of the micropore (patent document 1). Because the diameter of the micropore is small, a DNA molecule passing through the micropore passes through the vicinity of the gate portion. Since the DNA molecule has a negative charger the DNA molecule can be detected by the field-effect transistor. The distance between adjacent bases in the DNA molecule is 0.34 nm; thus, according to this method, it is difficult to analyze the base sequence by individually identifying the adjacent bases. Moreover, the document is silent as to the elongation reaction caused by addition of enzymes and substrates.
Hitachi discloses a DNA sensor that detects a target DNA using a field-effect transistor having a channel having a thin-line shape, in which a nucleic acid probe is immobilized on an insulating film surface on the channel and the change in conductivity of the channel caused by allowing a target DNA chain to flow along the thin-line channel is measured (patent document 2). This technology is also of a kind that detects a signal based on complementary binding of DNAs; thus, the base sequence cannot be analyzed. This known example also is silent as to elongation reaction caused by addition of enzymes and substrates.
Patent document 1: PCT Japanese Translation Patent Publication No. 2003-533676
Patent document 2: Japanese Unexamined Patent Application Publication No. 8-278281
Nonpatent document 1: Nature Biotechnology, vol. 16 (1998), p. 27, p. 40
Nonpatent document 2: Analytical Chemistry, 72 (2000) 1334
Nonpatent document 3: Intervirology, 43 (2000) 124-127
Nonpatent document 4: J. Phys. Chem. B 101 (1997) 2980-2985
Nonpatent document 5: Y. Shi et al., Anal. Chem., 71 (1999)
Nonpatent document 6: Ronagi, M.; Uhlen, M.; Nyren, P., Science, 1998, 281, 363-365